1. Field of the Invention
This invention pertains to tests for monitoring mutagenic agents and to transgenic animals. More specifically, the invention relates to genotypic changes in live animals that are monitored by the creation and use of transgenic non-human animals carrying marker DNA sequences that can be quickly recovered and examined for mutations.
2. Description of Related Art
Various agents, such as radiation, ultraviolet light, synthetic chemicals, natural substances, and aberrations in genetic replication and repair can produce mutations in DNA. Whenever a new chemical, drug, or food additive, for example, is to be taken from the laboratory to the marketplace, it must be tested for its toxicity and cancer-causing potential. Existing tests that assess the mutagenic potential of substances focus either on alterations of DNA in cultured cells or alterations in the health of test animals. Unfortunately, few tests that monitor alterations in DNA actually expose live animals to the agent to be tested. This is because it is very difficult to rapidly monitor small alterations in the genetic code simultaneously in many different organs. Tests to detect these mutations must be very sensitive. They must be able to detect a single mutation amongst millions of normal genetic units. The difficulty of this task currently makes this approach prohibitively expensive for live animal studies. Therefore, most current live animal toxicity tests use disease formation or large scale chromosomal alterations as an assay for gene alteration.
The problem of detecting small scale DNA alterations that are caused by potential mutagenic agents has generally been approached by performing studies on cells in culture (in vitro tests). The well-known Ame's test uses a special strain of bacteria to detect these mutation. Ames, et al., An Improved Bacterial Test System for the Detection and Classification of Mutagens and Carcinogens, Proc. Nat. Acad. Sci. 70:782-86 (1973). This test and many analogues that use other types of bacterial or animal cells permit the rapid screening of very large numbers of cells for the appearance of an altered phenotype. The appearance of this altered phenotypic trait reflects the occurrence of a mutation within the test gene. These tests are, however, insensitive to or nonspecific for many mutagens that result from metabolic activation of the agent being screened. Although attempts have been made to increase their sensitivity and specificity by activation of such metabolites with liver and other extracts it is noted that, for instance, the metabolites produced by these extracts are often not present at the same concentration as in the live tissues of an animal. Metabolites that are only produced in other organs are not detected at all.
Eukaryotic cell lines have also been used to detect mutations. E.g., Glazer et al., Detection and Analysis of UV-induced Mutations in Mammalian Cell DNA using Lambda Phage Shuttle Vector., Proc. Natl. Acad. Sci. USA 83:1041-1044 (1986). In this test a target test gene, the amber suppressor tyrosine tRNA gene of E. coli in a bacteriophage shuttle vector, was integrated into a genomic host mammalian cell line by DNA transfection. After exposing the host cell line to putative mutagenic agents, test genes were re-isolated, propagated in bacteria, and analyzed for mutations. Because the host is only a mammalian cell line and not a live animal, the test is incapable of accurately monitoring mutagenic metabolites of the agent being tested that are only produced at the appropriate concentrations by differentiated cells or the tissue of live animals.
Such test genes and large scale screening assays are not available for live animal studies. Short of relying on longterm animal studies that detect phenotypic changes that require a long time to be identifiable, such as tumors, organ failure, etc., current tests do not provide a means for monitoring organ-specific mutations of DNA. Hence, there exists a need for a system that places a test DNA sequence within an animal and is subsequently assayed on a large scale for mutations. There also exists a need for a test that detects mutations caused by chemical metabolites of the agent being tested. To be most effective the system needs to be capable of monitoring small genetic changes in as many tissues of an animal and as easily, rapidly, and inexpensively as possible.
The present invention produces a test that satisfies these needs. The test is a sensitive screen for the mutagenicity of all agents. The test not only monitors the mutagenic effects of the agent being screened it also monitors the mutagenic effects of all metabolites that occur in animal tissues of the agent being tested. It permits the identification of the nature of the mutation: e.g., DNA transition, transversion, deletion, or a point or frameshift mutation. The test is rapid and it is inexpensive relative to other tests. And, it will spot a potential mutagen rapidly before other more expensive tests can be completed.